Therapeutic proteins, or biologics, represent a $100 billion market that includes drugs such as antibodies, hormones, and many others. The clinical efficacy of biologics is critically determined by their circulating half-lives. Hence, various methods have been developed to increase their circulating half-lives by reducing clearance rates. This is commonly achieved by chemically conjugating biologics with biocompatible polymers in vitro. However, chemical conjugation is expensive, complicated, and often results in substantial losses of specific activity as well as a heterogeneous product mixture. These serious drawbacks have created a demand for a technology that can add biocompatible polymers to biologics without in vitro chemistry. To meet this demand, GlycoBac proposes a new, innovative method to add polysialic acid to biologics during their biosynthesis. Polysialic acid is (PSA) naturally found in the human body, and is a fully biocompatible, biodegradable and non-immunogenic polymer. In vitro chemically polysialylated biologics have already shown improved tolerance and pharmacokinetics compared to parent drugs. Moreover, sialic acid biology is well-understood through over half a century of research. Thus, PSA is an excellent choice to add to biologics with the goal of increasing their half-lives. Our new method uses existing N-glycans on glycoprotein biologics as a scaffold for PSA addition. Cells used for biologic production already add N-glycans to well-defined positions. We propose to enzymatically add PSA to these pre-existing N-glycans during biologic biosynthesis (in vivo). In contrast to chemical conjugation, our method is site-specific, does not require additional processing steps, and does not introduce additional cost and complexity. This SBIR project is designed to prove the feasibility of in vivo polysialylation as a next- generation platform technology. We will achieve this by Aims focused on producing a prototype cell line with a polysialylation pathway. These cells will be used to produce two polysialylated, commercially relevant glycoprotein biologics. For Phase I, we will use glycoengineered insect cells, as GlycoBac has extensive experience with this cell type. Our polysialylation technology is also compatible with mammalian cell lines such as CHO and PerC.6, which are commonly used to produce biologics. Phase I success will set the stage for a larger Phase II project focused on demonstrating the pharmacokinetics and activity of in vivo polysialylated biologics. Phase III commercialization of our in vivo polysialylation technology with private-sector partners is expected to significantly impact human health by enabling production of more efficacious glycoprotein biologics that require less-frequent dosing and/or reduced dosages.